Project Summary One in every 10 Americans is diagnosed with diabetes, and 90% of them have type 2 diabetes (T2D) due to insulin resistance and beta cell failure. Prevention and treatment of T2D remain an unmet medical need. The liver produces glucose during fasting to maintain normal blood glucose levels. This process is dysregulated in T2D, when resistance to or lack of insulin increases glucose production, resulting in hyperglycemia and its complications. Insulin regulation of hepatocyte glucose production (HGP) requires rapid changes in gene expression. FoxO1 plays an essential role in the transcriptional regulation of HGP. FoxO1 ablation impairs, but does not completely abolish the regulation of glucose production by insulin, likely due to incomplete suppression of phosphoenolpyruvate carboxykinase (Pck1) gene expression. These data highlight the heterogeneity of transcription factors (TFs) in regulating glucose production. The PI has recently identified TOX High Mobility Group Box Family Member 4 (TOX4) as a novel insulin-regulated TF, using DNA affinity purification from the Pck1 promoter and mass spectrometry (MS) in primary hepatocytes and liver samples. However, the metabolic function of TOX4 is unknown. In preliminary data, she shows that TOX4 is a transcriptional activator of Pck1 and glucose-6-phosphatase (G6pc). Knockdown of Tox4 in primary hepatocytes reduces glucose production by repressing these two genes. Moreover, in vivo KD of Tox4 in liver using adenovirus-encoded shRNA significantly inhibits gluconeogenesis and improves glucose tolerance and insulin sensitivity in C57BL/6J mice, while simultaneously promoting lipogenesis. These changes are more pronounced in diet-induced-obese (DIO) and diabetic db/db mice. Therefore, she hypothesizes that TOX4 regulates glucose and lipid metabolism in response to hormones. In this application, she will generate mice to ablate TOX4 in hepatocytes and critically investigate the function of TOX4 in hepatic glucose and lipid metabolism (Aim 1). She will further examine the mechanisms underlying hepatic TOX4 activation and regulation of its downstream targets (Aim 2). Successful completion of this application will illustrate the role of a new TF in regulating glucose and lipid metabolism in the liver in response to insulin. More importantly, these proposed studies will determine whether TOX4 is a potential therapeutic target in the treatment of insulin resistance and T2D.